characterization of bacteria isolated from cobia (rachycentron canadum) cultured in nha trang bay, vietnam

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES
Department of Aquatic Pathology



CHARACTERIZATION OF BACTERIA ISOLATED
FROM COBIA (Rachycentron canadum) CULTURED IN
NHA TRANG BAY, VIETNAM


By
VO LE THANH TRUC


A thesis submitted in partial fulfillment of the requirements for
the degree of Bachelor of Aquaculture





Can Tho, December 2013




CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES

Department of Aquatic Pathology


CHARACTERIZATION OF BACTERIA ISOLATED
FROM COBIA (Rachycentron canadum) CULTURED IN
NHA TRANG BAY, VIETNAM

By
VO LE THANH TRUC

A thesis submitted in partial fulfillment of the requirements for
the degree of Bachelor of Aquaculture


Supervisor
Ass. Prof. DANG THI HOANG OANH


Can Tho, December 2013
i

ACKNOWLEDGEMENT
First of all, the author thanks her supervisor, Ass. Prof. Dang Thi Hoang Oanh
for her invaluable supporting, guiding and encouraging during the research.
Many thanks also give to other doctors and masters of college of aquaculture and
fisheries, especially to members in department of aquatic pathology for kindly
help and support good conditions for working and learning.
The author would like to express her sincere to Ms. Tran Viet Tien, Ms. Bui Thi
Diem My, Ms. Au Thi Kim Ngoc, Mr. Le Thanh Can and Nguyen Trong Nghia
for their kindly help throughout the experimental period.

Last but not least, the author wants to give many thanks to her academic advisers
Mrs. Duong Thuy Yen, who always guiding and give useful advice during 4
academic years.
The author,
Vo Le Thanh Truc


ii

ABSTRACT
The purpose of this study was to isolate and characterize bacterial isolates which
were recovered from diseased Cobia cultured in Nha Trang Bay, Khanh Hoa
province. A total of eights diseased pathogens which were collected in October,
2012. Diseased fish displayed lethargic, hemorrhagic and whitish granules in
internal organs. Bacterial isolates were examined for morphology, selected
biochemical characteristics as well as susceptibility to common used antibiotics
in aquaculture. These isolates were identified as P. damselae subsp. Piscicida by
using API 20E test kit. Result of antibiotic sensitivity tests showed that there
were two out of eight isolates were resistant to doxycyline and tetracycline. All
isolates were completely susceptible to ampiciline, bicomarin and amoxyciline.
Enrofloxacine and ciprofloxacin are the antibiotics that all of the isolates were
susceptible with the largest zone. In another hand, eight isolates was susceptible
to erythromycin and Trim/sulfa with the intermediate inhibition zone range from
12 - 15mm.

iii

TABLE OF CONTENTS
ACKNOWLEDGEMENT i
ABSTRACT ii

LIST OF TABLES v
LIST OF FIGURES vi
CHAPTER 1 1
INTRODUCTION 1
1.1 G
ENERAL INTRODUCTION
1
1.2 R
ESEARCH OBJECTIVES
2
1.3 R
ESEARCH ACTIVITIES
2
CHAPTER 2 3
LITERATURE REVIEW 3
2.1

C
OBIA
(R
ACHYCENTRON CANADUM
)
AND
A
QUACULTURE OF COBIA IN
V
IETNAM
3
2.2

S
OME COMMON DISEASES IN
R
ACHYCENTRON CANADUM
(L
INNAEUS
,

1766) . 6
2.2.1 Streptococcal infection 7
2.2.2 Vibriosis 7
2.2.3 Photobacterium damselae sbsp. piscicida 8
2.3

S
OME COMMON ANTIBIOTICS USE IN AQUACULTURE
8
2.3.1 Oxytetracycline 8
2.3.2Beta-lactams 8
2.3.3 Florfenicol 9
CHAPTER 3 11
3.1

T
IME AND PLACES
11
3.2

M
ATERIALS

11
3.3

M
ETHODS
11
3.3.1 Bacterial isolation 11
3.3.2 Bacterial identification 11
3.3.3 Antibiotic susceptibility test 12
3.4

D
ATA COLLECTION
,
CALCULATION AND ANALYSIS
13
CHAPTER 4 14
iv

RESULTS AND DISCUSSION 14
4.1

F
ISH SAMPLING AND CLINICAL SIGNS
14
4.2

B
ACTERIAL IDENTIFICATION
15

4.2.1 Morphological observation 15
4.2.2 Physiological and biochemical characterization 16
4.2.3 Bacterial pathogen analysis by API 20E test kit ( BioMrieux, France) 16
4.3

A
NTIBIOTIC SENSITIVITY TEST
18
CHAPTER 5 21
CONCLUSIONS AND RECOMMENDATIONS 21
5.1 Conclusions 21
5.2 Recommendations 21
REFERENCES 22
APPENDIX 27


v

LIST OF TABLES
Table 4.1. Morphological, physical and biochemical characterization of the eight
isolates of bacteria from diseased cobia samples.
Table 4.2. Result from API 20E biochemical test strip.
Table 4.3. Sensitivity of bacterial isolates to different antibacterial drugs (data
are the diameters of the antibacterial area in the plate, the unit is mm).

vi

LIST OF FIGURES
Figure 4.1. Lethargic fish.

Figure 4.2. Hemorrhage in body cavity and white spot on internal organs.
.Figure 4.3 A Morphology of isolated bacteria on NA
+
.
Figure 4.3 B Gram stain of isolated bacteria.
Figure 4.4 O/F test.

Figure 4.5 API 20E test result for P. damselae sub sp. Piscicida after 24hours
incubation.
Figure 4.6 Antibiotic sensitivity test.
1


CHAPTER 1
INTRODUCTION
1.1 General introduction
Cobia, Rachycentron canadum, is considered one of the most suitable candidates for
warm, open-water of aquaculture in the world. They contribute widely in warm marine
water to tropical waters of the West and East Atlantic, through Caribbean and Indo-
Pacific region. Cobia have good traits, most important traits are growing rapidly and
high flex quality. They reach harvest sizes (6kg - 8kg) after one and half years (FAO,
2009). Furthermore, they can tolerate in wide range of salinities (5ppt – 44.5ppt) and
temperatures (1.6
0
C – 32.2
0
C). Aquaculture research with cobia was first reported in
1975 but there were any large-scale commercial production of cobia until 2006. With
the production report to FAO in 2004, China and Taiwan are two main production
countries of cobia over the world. In Vietnam, farming of cobia is a new species to

aquaculture, which are cultured by small-scale to medium-scale family farms as well as
cooperative farms. The main factor constraining development of cobia culture in
Vietnam is a shortage of quality fingerlings, although hatchery production in Vietnam is
increasing at a rapid rate (Nhu et al., 2010)
Like other species, cobia also gets diseases during culture either ponds, tanks or
cages. Managing disease and parasite issues has been identified, particularly by the
Taiwanese, to be one of the major challenges with regard to cobia culture so far (FAO,
2013). Bacteria cause diseases by some clinical signs: whitish, granulomatous deposits
on kidney, liver and spleen, ascites in peritoneal cavity.
Disease during culturing of cobia is one of the most concerns of farmer and
producer because of high mortality. Cobia is the new species for aquaculture not only in
Vietnam but also in the world. So, this thesis: “Characterization of bacteria isolated
from cobia (Rachycentron canadum) (Linaeus, 1766) cultured in Nha Trang Bay,
Vietnam” was conducted to update information of diseases in other to identify common
bacterial pathogens in cukturedcobia. Hence, recommending treatments and antibiotics,
increasing productivity, profitability for farmers.






2



1.2 Research objectives
The research is aimed to identify isolated bacteria form diseased Cobia cultured
in Nha Trang Bay, Vietnam and their susceptibility to commonly used antibiotics.
1.3 Research activities

1. The first activity is to identification of isolated bacteria to species level to find
out which bacterial pathogen caused diseases on cobia.
2. The second activity is to do antibiotic sensitivity tests which aim to figure out
the susceptibility to common used antibiotics.




















3



CHAPTER 2
LITERATURE REVIEW

2.1 Cobia (Rachycentron canadum) and Aquaculture of cobia in Vietnam
Rachycentron canadum, commonly is a marine finfish species with high potential for
aquaculture in Vietnam and all over the world. They are also called black kingfish, is a
fast growing pelagic fish found in the tropical/subtropical seas all over the world. Cobia
has large body with a maximum length of 2 meters and maximum weight of 60
kilograms. After spawning, larvae need 24 hours of fertilization for hatching in high
salinity water and juveniles usually swim to warmer offshore water. Cobia broodstocks
are captured by professional fishermen and transported to hatcheries. Larvae and
juveniles are stocked in ponds or tanks and grow-out one are cultured in open ocean
cages. Sets in clear water, far away from industrial zones, vessels and water current of
0.2 – 0.6m/s are suitable for cage construction as well as salinity from 27ppt to 33ppt
and dissolve oxygen are 4mg/L to 6mg/L for growth out culture. Production, while still
small, has increased significantly over the past three years. Most production currently
comes from China and Taiwan Province of China and totaled around 20,000 tonnes in
2003 (FAO, 2006). Production of this fast-growing (to 6 kg in the first year) species is
set to expand rapidly, not only in Asia but also in the Americas. Cobia fingerlings used
for aquaculture are mainly hatchery produced, with Taiwan Province of China being one
of the first to establish hatchery production. Seed production in 1999 was around three
million fingerlings of about 10 cm with a market value of US$0.50 per fish. Due to the
rapid growth of cobia and its suitability for commercial production, cobia aquaculture
has become more and more popular. To date, research and development of cobia
aquaculture has been initiated in over 23 countries and territories, half of them in the
Asian-Pacific region. Statistics of FAO (2009) show that the global aquaculture
production of cobia has been increasing rapidly from only 9 tons in 1997 to nearly
30,000 tons in 2007. Meanwhile, the volume from capture fisheries has remained stable,
around 10,000 tons annually. It is estimated by the authors that in 2008, Vietnam has
produced 1500 tons, thus, being the third largest cobia producer in the world.
The cobia, Rachycentron canadum, is an important marine fish first artificially
propagated and cultured in Taiwan for one decade, especially in sea-cage farming (Ku

4

and Lu 2000, Liu et al. 2003b). The farming of cobia will presumably become an
emerging aquaculture industry in the near future since the industry is now being carried
out in some tropical and subtropical regions of the Far East such as Hainan, China,
Okinawa, Japan and Viet Nam. However, the industry faces various threats including
viral, bacterial and parasitic diseases (Ku and Lu 2000, Liu et al. 2003b). History of
cobia aquaculture in Vietnam dates back to 1997 when research on cobia reproduction
started, leading to the first successful production of about 12,000 fingerlings in 1999 at a
marine hatchery located in Cat Ba Island, Hai Phong province (Van Can Nhu, 2010).
Marine fish species are common in marine cages and ponds in Viet Nam’s coastal water,
including cobia, which is increasingly popular in the north (from Vinh Ha Long Bay and
Bai Tu Long Bay), centre (Van Phong Bay, Khanh Hoa) and also beginning to be
cultured in the south-central provinces such as Ba Ria – Vung Tau and Kien Giang.
Marine fish in Viet Nam are grown in cages and ponds. The farms tend to be small
family-owned operations, although industrial-scale developments are also starting.
Marine aquaculture gradually develops with expanding farming of this potential species,
opens up a new direction for offshore and inland aquaculture. In 2002, the first
commercial batches of more than 20,000 cobia fingerlings were produced under a
project co-funded by the Government of Vietnam and the Government of Norway. Since
then, research on improvement of larvi-culture technology of cobia has resulted in better
growth, survival and production. During 2008, more than 400,000fingerlings were
produced at a hatchery of the Research Institute for Aquaculture No1 (RIA-1) at Cat Ba
Island in addition to a smaller number produced at a private hatchery at Khanh Hoa
province. Production in Vietnam was estimated to be 2,600 tonnes in 2009 (Nhu et al,
2010).
The main factor constraining development of cobia culture in Viet Nam is shortage of
quality fingerlings although hatchery production in Vietnam is increasing at a
rapid rate (Nhu et al, 2010). The cobia is the only emerging tropical mariculture
species for which the life cycle has been fully closed and fingerling availability is not

limiting factor (Nhu, 2005). For example, the Research Institute for Aquaculture No1 in
Vietnam produced 400,000 fingerlings in 2007 and 900,000 in 2008 (Nhu et al, 2010).
The industry still relies on fingerling imports from Taiwan and China (Hainan) (Huy,
2008). Earlier farming of cobia–mainly in the southern Vung Tau region depended on
imported fingerlings, but the more stable fingerling availability has now led to several
larger fish farms to grow cobia–the Norwegian financed Marine Farms Vietnam being

5

the largest. The production during 2009 from the latter company could reach 1000
tonnes.
Cobia farming in Vietnam was initially conducted in simple, small scale wooden raft
cages installed in closed bays, using wild-captured fingerlings as seed and trash fish as
feed. The hatchery-fingerlings were imported from Taiwan or China before 2002 when
the locally produced fingerlings were available. In a 2005 survey, there was a total of
16,319 marine cages producing approximately 3510 tons of marine aquaculture products
(Ministry of Fisheries and The World Bank, 2006). The main constraint of cobia
farming in Vietnam is market development. In addition, insecurity in supply of high
quality juveniles and then some geographical or climatic constraints such as low
temperature during winter in the North, and tropical typhoons occurring especially in
autumn in central Vietnam. The main grow-out constraints would be parasites, bacteria
and virus and feed quality and management to keep the FCR low. Quality and quantity
of cobia fingerlings affect the profit of cobia farming (Miao et al., 2009). At present,
cobia fingerlings in Vietnam are produced mainly in the semi-intensive systems.
Although this rearing method is relatively simple, low-cost and easy-copied, there are
some uncontrolled factors and it has been experienced to result in relatively low
survivals (Benetti et al., 2007; Liao et al., 2004; Weirich et al., 2004) and the cobia
fingerlings obtained from these systems have been reported to be of unstable quality i.e.
potential of size variation and parasite infection. Thus, the intensive production needs to
be developed at appropriate proportion to reduce risk and to ensure sustainable

development. However, the present intensive rearing method is relatively expensive and
sophisticated and there is a need to simplify the protocol to reduce the production costs.
In this regards, to shorten the live prey feeding period, improve nutritional condition and
hatchery zoo-techniques will be elements to improve growth, survival and quality of
cobiafingerlings.
Disease outbreak is another challenge for sustainable development of cobia aquaculture
in Vietnam. During larval rearing, infections of protozoa such as Vorticella sp., Epistylis
sp., Pseudorhabdosynochus epinepheli, Benedenia and Trichodina have been detected
(Le and Svennevig, 2005). Samples collected from sudden crashes of cobia larviculture
in RIA-1's hatcheries revealed Viral Nervous Necrosis (VNN) infection of 20–30% (Le
and Svennevig, 2005). The vertical transmission of VNN has been confirmed. The use
of iodine and peroxide did not effectively eliminate VNN from fertilized eggs (Le and
Svennevig, 2006). Therefore, quarantine and screening of the broodstock before the
reproduction cycle is essential to prevent the VNN vertical transmission. In addition,

6

high mortality caused by Amyloodinium ocellatum attaching to gills and skin of cobia
juveniles has been detected in RIA-1's hatcheries in 2005 and 2006. A high density of A.
ocellatum in gills of cobia juveniles might inhibit breathing, leading to slow movement
and finally cause high mortality. Formalin treatment at a concentration of 0.03–0.1 mL
L−1 for 1 h with strong aeration or fresh water treatment can be effective in case the
first symptom is detected in time. It is also important to mention the challenge of
tropical typhoons in the areas. Vietnam is located in south-east Asia, situated in the
western Pacific Rim exposed to the tropical typhoons from the Pacific Ocean during
autumn. Large-scale farms need to be situated in fairly open sea areas to maximize the
production, but can also be exposed to harsh weather conditions. The failure of some
cobia farms in the Northern central region during a typhoon in 2005 showed to be
caused by insufficient dimensioning of the mooring system. At the moment, the
Government of Vietnam are supporting development of new semi-submersible cages

(National project KC07/03-06/10), which can be controlled to sink temporarily to avoid
surface damages during stormy conditions. This cage type can be installed in semi-open
or open sea areas where a larger exposure resulting in high of water exchange will
contribute to the maintenance of good water quality. Alternative grow-out systems such
as land-based recirculation systems should also be considered to provide more options
for the industry in regions haunted by typhoons.
2.2 Some common diseases in Rachycentron canadum (Linnaeus, 1766)
Bacteria and parasites can cause serious sub-lethal impacts on production by
reducing the growth and feeding efficiency of stocks as well as leaving fish more
susceptible to other infections (Mustafa et al, 2001; Boxaspen, 2006; McLean et al,
2008). The reported bacterial diseases of cobia include mycobacteriosis, vibriosis,
pasteurellosis, and streptococcosis, which are caused by pathogens including
Mycobacterium marinum, Vibrio anguillarum and V. ordalii, Pasteurella piscicida and
Streptococcus spp. (Liao et al., 2004, Lowery and Smith, 2006). The viral disease
lymphocystis, and the parasitic diseases myxosporidosis, Trichodina spp., Neobenedenia
spp., and Amylodinium spp. also can affect cobia (Kaiser and Holt, 2005). To the
present, most study in cobia diseases focus on parasitic pathogens. In Taiwan, scientists
are given some common bacterial pathogens that make high mortality up to 80% or
more in young cobia in culture cobia (Lin et al., 2006).



7

2.2.1 Streptococcosis
Streptococcal infection of fish is considered as re-emerging disease affecting a variety of
wild and cultured fish throughout the world. Five different species are considered to be
of significance as fish pathogens: Lactococcus garvieae, L. piscium, Streptococcus
iniae, S. agalactiae, S. parauberisand, Vagococcus salmoninarum. Therefore,
streptococcosis of fish should be regarded as a complex of similar diseases caused by

different genera and species capable of inducing a central nervous damage characterized
by supperative exophthalmia and meningoencephalitis. Warm water streptococcosis
typically involves L. garvieae, S. iniae, S.agalactiaeanm, S. parauberis. It is important
to report that the etiological agents of warm water streptococcosis are considered also as
potential zoonotic agents capable to cause disease in humans. Among these fish
streptococci, L. garvieae, S. iniaeand S. parauberis can be regarded as the main
etiological agents causing diseases in marine aquaculture in both nursery and grow-out
culture stages.
2.2.2 Vibriosis
Vibriosis is a disease characterized by haemorrhagic septicaemia and caused by various
species of Vibrio. It occurs in cultured and wild marine fish in salt or brackish water,
particularly in shallow waters during late summer. Within the Vibrionaceae, this genus
includes the human pathogens V. cholerae, V. mimicus, V. parahaemolyticus, and V.
vulnificus, as well as fish pathogens Listonella anguillarum(formerly V. anguillarum),
V. ordalii, V. damsela, V. carchariae, V. vulnificus, V. alginolyticus, and V. salmonicida
(Reed and Francis-Floyd, 2002). Vibrio spp. pathogens also affect other species of
marine fish, penaeid shrimp, as well as abalone (Liu et al., 2004). In addition, Vibrio
spp. bacteria account for a significant portion of the food-borne infections from eating
raw or undercooked shellfish (Thompson et al., 2004) and become the economically
most important disease in marine fish culture, affecting a large number of species. It is
also an important disease of many wild fish populations. Fish affected by vibriosis show
typical signs of a generalized septicemia with hemorrhage on the base of fins, ulcers on
body surface, swelling and boils, exophthalmia and corneal opacity. Moribund fish are
frequently anorexic with pale gills, which reflect a severe anaemia. Oedematous lesions,
predominantly centered on the hypodermis, are often observed. On the top of the boils,
the epidermis is destroyed and the skin is greyish white. Around the boil, the skin is
hemorrhaged. Internally there are hemorrhage in liver and intestine, and there is fluid in
the heart lumen. Histologically, the muscle fibres are widely separated.

8

2.2.3 Pasteurellosis
Photobacterium damselae ssp. piscicida (Gauthier et al. 1995) is a Gram-negative, non-
motile, bipolar coccobacillus (Snieszko et al. 1964), which was previously known as
Pasteurella piscicida (Snieszko et al. 1964). It is the causative agent of the fish disease
photobacteriosis, also known as pasteurellosis or pseudotuberculosis. High mortalities of
P. piscicida infection were first observed in natural populations of white perch (Morone
americanus) and striped bass (Morone saxatilis) in 1963 in Chesapeake Bay (USA)
(Snieszko et al, 1964), and it has caused economic loss to the fish farming industries in
Japan (Kusuda and Yamaoka, 1972). Several countries in the Mediterranean area
(Toranzo et al, 1991; Bakopoulos et al, 1995, 1997; Baptista et al, 1996; Candan et al,
1996; Topic Popovic et al, 2001) have encountered similar high mortalities in cultured
sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). The pathogen has also
been isolated and caused high mortalities in farmed cobia (Rachycentron canadum) in
Taiwan 2001 (Lopez et al, 2002).
Photobacterium damselae ssp. damselae (Smith et al. 1991; Truper and De Clari 1997)
which was formerly classified as Vibrio damsela is a halophilic bacterium causing skin
ulcers in warm and cold water fish (Love et al. 1981; Sakata et al, 1989; Fouz et al,
1992a,b).
2.3 Some common antibiotics use in aquaculture
2.3.1 Oxytetracycline
Oxytetracyline is also called tetracycline antibiotic which used to treat infections
with bacteria in quaculture farms such as chlamydia, mycoplasma, protozoa and several
ricketsiae. In other words, oxytetracyline is a broad spectrum antibiotic that is active
against a wide variety of bacteria. However, resistance has been acquired by coliforms,
mycoplasma, streptococci and staphylococci (Bui Kim Tung, 2001).
2.3.2Beta-lactams
This group includes Amoxicillin and Ampicillin. Amoxicillin is active against
penicillin-sensitive Gram-positive bacteria and some Gram-negative bacteria. Gram-

positive spectrum includes alpha- and beta- haemolytic Streptococci, some
Staphylococci species and Clostridia species. Gram-negatives: Escherichia coli, many
strains of Salmonell, and Pasteurellamultocida are susceptible to destruction by beta-

9

lactamases. Ampicillin is active against alpha- and beta- haemolytic streptococci,,
including Streptococcus equi, non-penicillinase-producing Staphylococcus species, and
most trains of Clostridia. Also effective against Gram-negative bacteria, such as
Escherichia coli, Salmonella and Pasteurellamultocida (Bui Kim Tung, 2001).
2.3.3 Florfenicol
This fluorinated antibiotic, derived from thiamphenicol, is a potent and broadly acting
bacteriostatic agent. Activity similar tochloramphenicol, including many Gram-positives
and Gramnegatives and without the risk of inducing human aplastic anaemia associated
with chloramphenicol. It is effective in the treatment of infections caused by Pasteurella
piscicida, Aeromonas salmonicida, Vibrio anguillarum, and Edwardsiella tarda.
Pharmacokinetically, florfenicol use has been reported among some species of fish such
as Atlantic salmon (Salmosalar), in which a bioavailability of more than 95% is present,
exhibiting a good distribution among all of the organs and tissues. Its half-life in fish is
less than 15hrs (Yanong and Curtis, 2005).
2.3.4 Enrofloxacin
Enrofloxacin was developed as an antimicrobial agent during the 1980s for exclusive
use in veterinary medicine and has proven to be effective in the treatment of bacterial
diseases that affect aquaculture organisms. The mechanism of enrofloxacin acts at the
level of the cellular nucleus, inhibiting DNA synthesis. During the multiplication phase
of the bacteria, the DNA folds and unfolds alternately. This process is controlled by the
enzyme DNA gyrase, which is inhibited by enrofloxacin, causing a collapse of bacterial
metabolism and preventing the genetic information from being copied, thus causing the
bacteriocidal effect (Williams et al., 2002). The information related to this antibiotic for
the most widely grown shrimp species such as Litopenaeus vannamei is scarce, but

pharmacokinetic studies on enrofloxacin have been carried out using other species, such
as crab (Scylla serrata), tilapia (Oreochromis niloticus), black shrimp (Penaeus
monodon), Chinese shrimp (Penaeus chinensis), and European seabass (Dicentrarchus
labrax) (Intorre et al., 2000; Tu et al., 2008; Wen et al., 2007; Xu et al., 2006). It is
important to note that the pharmacokinetic results for enrofloxacin obtained for these
species should not be extrapolated to other aquatic species, because each organism
possesses a different metabolism, and the cultivation conditions may have a significant
influence over the kinetic behavior displayed by the antibiotic.

10


2.3.5 Ciprofloxacin
Ciprofloxacin is the main metabolite of Enrofloxacin and is active against a broad
spectrum of aerobic Gram (-) bacteria, including enteric pathogens such as
Pseudomonas and Serratia marcescens. It is also active against Gram (+) pathogens,
even when these bacteria have developed resistance to other antibiotics, such as
penicillin (Wen et al., 2007). It is not active against anaerobic bacteria and may be used
occasionally, in combination with other antibacterial agents, for the treatment of
mycobacterial infections. The antibacterial effects of ciprofloxacin arise from its
inhibition of Topoisomerase IV and bacterial DNA gyrase, which act by cleaving the
DNA of the bacterial chromosome and rejoining the ends once a superhelix is formed
(Banerjee et al., 2007). When these enzymes are inhibited, bacterial cell multiplication is
interrupted.

11

CHAPTER 3
RESEARCH METHODOLOGY
3.1 Time and places

- Time: The thesis was conducted from June to December, 2013.
- Place: Isolation, identification, PCR will be done at Department of Aquatic
Pathology - College of Aquaculture and Fisheries, Can Tho University.
3.2 Materials
- Equipment: Tissues paper, gloves, aluminum foils, nylon bags, refrigerators, petri
dishes, alcohol lamps, inoculating loops, 100 – 1000µl pipets, alcohol spray, API
test kit.
- Chemicals: absolute alcohol, NaCl, distilled water, antibiotic samples, glycerol,
crystal violet solution.
- Culture media: NA(Nutrient Agar) and TCBS (Thiosulfate Citrate Bile Salts
Sucrose) with addition 1.5% NaCl, OF (Oxidative – Fermentative media), Blood
Agar.
3.3 Methods
3.3.1 Bacterial isolation
Diseased juvenile cobia (R. canadum) were sampled from disease cages in Nha Trang
Bay, Vietnam during October, 2012. These fish displayed lethargic, whitish,
granulomatous deposits on kidney, liver, hemorrhage in peritoneal cavity.
Five samples were collected in each cage with the recording of health status, water
conditions and feeding. Ten strains were stored at 70°C in nutrient broth (Merck)
containing 15% glycerol and supplemented with 1.5 % sodium chloride.
3.3.2 Bacterial identification
After being cultured for 24 hours at 28°C, the uniform colonies were removed for
further culture. The isolates were purified after several times of culture and then
inoculated on test tube slants as pure culture and stored on 4°C for further analysis of
identification. Bacteria were inoculated in NA media for 24 – 48 hours. Shape and color
of colonies were observed and recorded. Cell morphology was studied in Gram-stained

12

preparations from nutrient agar (Himedia) plates supplemented with 1% NaCl according

to Hucker’s modification method (Barrow & Feltham, 1993). Bacterial identification
procedure in this thesis followed Manual for the isolation and identification of fish
bacterial pathogens (Frerichs and Millar, 1993).
Bacterial pathogens were identified follow basis biochemical tests: Gram staining,
motility, oxidase, catalase, hemolytic, API test. The specific steps for some of the
biochemical tests were described in Appendix. Strains of bacteria were identified using
API 20E test kit (BioMerieux, France) following the manufacture instruction.
3.3.3 Antibiotic susceptibility test
Antibiogram test was carried out by the method of Geert Huys (2002).
Bacterial cultivation and material preparations (DAY 1)
-
The organism to be tested was cultivated on TSA medium at 28
0
C under aerobic
atmosphere. Streak out the pure culture on TSA plate in a way that distinct colonies
were obtained.
-
Prepare the desired volume of TSA medium according to the manufacturer’s
instructions. Before pouring the agar medium, bottles cooled to 40-50
0
C in room
temperature. All plates were poured on a flat, horizontal surface to an identical
depth of 5 mm + 1 mm (corresponding to 20 mL + 1 mL of medium in 10 cm radius
petri dishes).
-
Prepare tubes containing 5 mL sterile NB (Nutrient Broth) solution (with 0.85%
NaCl)
Inoculation of the antibiogram (DAY 2)
-
Take a number of pure colonies from the fresh grown plate culture to suspend in a

tube containing NB solution until turbidity (visually) corresponding to 1.0
McFarland standard is reached. It remained important to take more than one colony
in order to obtain a representative sample.
-
Using a micropipet, spot 100 µL of the standardized suspension on the surface of
TSA plates. Spread plate the suspension using a sterile glass triangle rod. Allow to
dry the plates. Longer drying times allow pre-incubation of the cells which should be
avoided. Manually using sterile forceps applied the discs onto the agar surface. Discs
must not be relocated once they have made contact with the agar surface. Incubate
the plates at 28
0
C for 24 hours.

13

Reading of the antibiogram (DAY 3)
-
The diameter of the inhibition zones were measured to the nearest mm from the point
of abrupt inhibition of growth (using a callipers or mm ruler). If the plates were not
sufficiently grown, read again after 48 h incubation. Plates on which the growth of
the test strain produced isolated colonies (less than semi-confluent growth) should
not be read. If zones of inhibition produced by adjacent discs overlap to the extent
that two measurements at right angles cannot be made, the zones around these discs
should not be recorded. Equally, zones demonstrating significant distortion from
circular should not be reported (corresponding to 20 mL + 1 mL of medium in 10 cm
radius petri dishes).
3.4 Data collection, calculation and analysis
Data collection and analysis were done by using Microsoft Word and Microsoft Excel
2007.

14

CHAPTER 4
RESULTS AND DISCUSSION
4.1 Fish sampling and clinical signs
From each cage, fish were collected both healthy and diseased one. The infected fish vary
from no external indicators through had some gross signs which can see easily by snake
eyes such as lethargic, abnormal swimming and skin lesioning. Internally, compare to
normal fish, ascites may be present in the peritoneal cavity and the host liver and kidney
may pale in color while the spleen may have white tubercules present. Healthy cobia had
clear internal body cavity, whereas the diseased body cavity with hemorrhage. (Figure
4.1 and 4.2).
Figure 4.1. Lethargic fish.

Figure 4.2. Hemorrhage in body cavity and white spot on internal organs


15

Bacteria were isolated from brain, kidney and liver and streak on nutrient agar (Merck)
supplemented with 1.5% NaCl. Eight dominant bacterial isolates (isolate CB 2-2_N, CB
2-2_T, CB 2-2_G, CB 2-4_T, CB 2-4_N, CB 1-2_N, CB 1-4_G and CB 1-4_T) were
recovered from kidney, brain and liver of the cobia suffering from diseases.
4.2 Bacterial identification
4.2.1 Morphological observation
Bacterial isolates (CB 2-2_N, CB 2-2_T, CB 2-2_G, CB 2-4_T, CB 2-4_N, CB 1-2_N,
CB 1-4_G and CB 1-4_T) from diseased cobia displayed colonies on nutrient agar
supplemented with 1.5% NaCl

media with rounded, slightly convex, smooth and milky
color (Figure 4.3 A and B). After 24 hours of incubation, those strains presented in
green color on TCBS. Those isolates indicated the characteristics of Gram negative
bacteria, short rod shape, positive mobility, positive oxidase and catalase, they displayed
the ability of fermentation and oxidation of glucose.
Figure 4.3 (A) Morphology of isolated bacteria on NA
+
. (B) Gram stain of isolated
bacteria.

Figure 4.4 O/F test

A
B

16

Table4.1. Morphological, physical and biochemical characterization of the eight isolates
of bacteria from diseased cobia samples.
Items tested
Isolates
P. damselae
subsp.
Piscicida
(Magarinõs
et al., 1992)
CB 1-
2N
CB 1-
4T

CB 1-
4G
CB 2-
2N
CB 2-
2T
CB 2-
2G
CB 2-
4T
CB 2-
4N
Gram stain - - - - - - - - -
Morphology r r r r r r r r r
Color of
pigment
milky

milky

milky

milky

milky milky

milky

milky milky
Motility - - - - - - - - -

Oxidase
reaction
+ + + + + + + + +
Catadase
reaction
+ + + + + + + + +
Aerobic
fermentation

+ + + + + + + + +
Anaerobic
fermentation

+ + + + + + + + +
Gas
production
- - - - - - - - -
0/129
resistance
+ + + + + + + + +
Notes: ( r ): rod shape; ( - ): negative; ( + ): positive
4.2.2 Physiological and biochemical characterization
Isolates CB 2-2_N, CB 2-2_G, CB 2-2T, CB 2-4_T, CB 2-4_N, CB 1-2_N, CB 1-4_G
and CB 1-4_T were characterized as genus Pasteurellosis because all of them were
bipolar rod shape, positive for the oxidase, catadase reaction, non-motile. Moreover,
fermentation in O/F media also gave positive result without gas production and sensitive
to the vibrio static agent O/129. In the test with blood media, these isolates give the
result that they do not have the ability to dissolve blood after 24 hours incubation.
4.2.3 Bacterial pathogen analysis by API 20E test kit ( BioMrieux, France)
Identification by API 20E test kit aimed to identify correctly bacterial pathogens causing

diseases on cobia. A profile number of 201500457 obtained from API 20E system
corresponded to Photobacterium damselae sub sp. Piscicida

17

Table4.2. Result from API 20E biochemical test strip:
No.

Indicators

CB
1-2N
CB
1-4T

CB
1-4G

CB
2-2N

CB
2-2T
CB
2-2G

CB
2-4T
CB
2-4N

P. damselae
subsp.
Piscicida
(Magarinõs
et al., 1992)
1 ONPG - - - - - - - - -
2 ADH + + + + - + - + +
3 LDC - - - - - - - - -
4 ODC - - - - - - - - -
5 CIT - - - - - - - - -
6 H
2
S - - - - - - - - -
7 URE + + + + - + - + -
8 TDA - - - - - - - - -
9 IND - - - - - - - - -
10 VP + + + + + + + + +
11 GEL - - - - - - - - -
12 GLU + + + + + + + + +
13 MAN - - - - - - - - -
14 INO - - - - - - - - -
15 SOR - - - - - - - - -
16 RHA - - - - - - - - -
17 SAC - - - - - - - - -
18 MEL - - - - - - - - -
19 AMY - - - - - - - - -
20 ARA - - - - - - - - -

Note: ( - ): negative result; ( + ): positive result ; ONPG: β-galactosidase (o-nitrophenyl-β-

Dgalactopyranoside); ADH: arginine dihydrolase; LDC: lysine decarboxylase; ODC: ornithine
decarboxylase; CIT: citrate; H
2
S: hydrogen sulfide; URE: urease; TDA: tryptophane deaminase; IND:
indole; VP: Voges-Proskauer reaction; GEL: gelatin; GLU: glucose; MAN: mannitol; INO: inositol;
SOR: sorbitol; RHA: rhamnose; SAC: sucrose; MEL: melibiose; AMY: amygdaline; ARA: arabinnose

The results of eight isolates bacterial pathogen shows positive responses for arginine
dihydrolase (ADH), lipase activities , and is negative for indole (IND), nitrate reduction,
gelatinase (GEL), amylase (AMY), tryptophane deamynase (TDA) and hydrogen sulfide
(H
2
S) production as well as create β-galactosidase (ONPG). They also had negative